1 Field of the Invention
This invention relates to the areas of the biochemical and chemical analysis of molecules in cells, and in particular to an assay and method for measuring the activation of internal chemical activity of a plurality of proteins in a single cell, a population of cells, or portion of a cell.
2. Description of the Prior Art
All cells are composed of complex networks of proteins that allow them to respond to the environment and to go about the business of life (FIG. 1-2). At any given time all of the proteins in a cell are not active. The determination of which proteins are active in a living cell and how networks of proteins interact in a living cell is a complex problem in biology and in pharmaceutical development.
The ability of an organism to respond to a stimulus is essential for its survival. Disease often results from a breakdown in communication between and within cells. For example, type II diabetes results from the inability of cells to receive and/or process the insulin signal that regulates carbohydrate metabolism. Signals are generally received at the cell membrane by molecules called receptors. The binding of a signaling molecule to a receptor at the outside cell surface causes the receptor to change shape. This, in turn, modulates the chemical activity of the receptor on the inside of the cell. Any given cell will have a number of different receptors enabling it to respond to various signals. The type of tissue from which the cell comes determines which receptors are present on its surface. Different cells respond to different signals, and the same signal can elicit a different response in cells that come from different tissues. For example, epinephrine causes the contraction of vascular smooth muscle but the relaxation of intestinal smooth muscle. How a cell responds to a signal is a result of which proteins are present and activated in the cell at the time of and after the signal is received.
In response to a signal, various proteins in the cell are activated and deactivated. A common strategy used by cells to change the activity of a protein is to add or remove a phosphate group to or from the protein. The addition of a phosphate group to a protein is performed by enzymes (which are themselves proteins) called kinases. Many of the proteins in the above described networks are enzymes. In addition to kinases, many other types of enzymes also exist in cells. Phosphatases, nucleases, glycosidases, lipases and proteases are but a few examples. Enzymes are important drug targets. A challenge in developing drugs against kinases and other types of enzymes is to find molecules that are specific for only the enzyme of interest. Many potential drugs may have a desired effect on its expected target, but may also have an undesired effect on the activity of one or more additional proteins within the cell. Presently available in vitro biochemical assays for enzyme activity often give misleading results since other enzymes and modulators of enzyme activity present in a living cell are not present in the assay (Table I). These factors are difficult to add to an assay because their identity and concentration are often unknown. The situation is further complicated by the fact that the concentration of these factors and the activity of the proteins can change over time (Table II). In order to accurately study the full effect of a drug, all of the components of the signaling pathways must be present and intact.
Thus, it is highly desirable to be able to analyze the activity of a protein in its native cellular environment. Additionally, since the proteins exist in interconnected networks, the activities of many proteins will be affected by perturbations in the cellular environment or by the abnormal activity of another protein within the networks. Thus, it is important to be able to measure the activities of a plurality of proteins at the same time in the cell(s) of interest. We are not aware of any biochemical assay for protein activity capable of measuring the activities of multiple proteins simultaneously in living cells except that described below.
It is now known that many disease states are related to inappropriate protein activity, either too much or too little activity. An example is the human cancer chronic myelogenous leukemia (CML). In most cases of CML a kinase, the protein product of the oncogene bcr-abl, is present in an inappropriately “turned-on” state. This inappropriate activation leads to the uncontrolled growth of blood cells that is manifested as cancer. Other protein products of oncogenes are known to play a role in the development of cancer. However, the presence of the gene or its protein product is not perfectly correlated with the appearance of cancer. A means to measure the activity of such proteins and that of normal proteins in the same cell with the relationships of the protein pathways intact will reveal important insights into disease processes. A profile of a disease composed of a map of the active and inactive proteins in affected cells can be expected to provide a more accurate understanding of the molecular pathogenesis of the disease than even that revealed by current genomic and proteomic techniques (Table III). A database of protein activity in the cells of different tissues, in healthy and diseased cells, and in cells responding to different environmental and pharmacolgic stimuli would have a dramatic impact on biomedical research, pharmaceutical research, and even on our basic understanding of the natural processes of all living organisms.
Existing techniques for the measurement of enzyme activation in a single cell, group of cells, or a portion of a cell have inherent limitations. In recent years much has been made of proteomics, the identification of all proteins produced by an organism, as a means for extending our knowledge beyond the genomics revolution. Unfortunately, current proteomic technologies have significant shortcomings in the study of enzyme function. For the past two decades, the gold standard has been two dimensional-gel electrophoresis. This technique gives a very high resolution for protein separations, but it is difficult to perform, and it cannot detect many important cellular proteins, especially those in low abundance or those with a hydrophobic character (traits typical of many if not most enzymes). The new mass spectrometry techniques combined with bioinformatics have improved the identification of proteins separated by electrophoresis techniques, but do not solve the fundamental issues of performance difficulty and sensitivity. The new chip-based methods hold the as yet unfulfilled promise for identifying large numbers of proteins quickly; however, sensitivity, specificity, and quantification are still issues to resolve. It is important to understand that nearly all current proteomic approaches strive to identify proteins, and in some cases to provide a rough quantitation of protein concentration. However, these approaches do not directly measure the critical parameter of the most crucial proteins: the activity of enzymes. Neither the presence or concentration of an enzyme is a valid measure of its activity. Attempts are made to measure a protein's activity by the identification of a phosphorylated species (many proteins are “switched on” by the addition of one or more phosphate groups), but such an approach can provide only an indirect measure of activity, and detecting such species in a single cell, group of cells, or a portion of a cell is fraught with difficulty.
Until recently, traditional biochemical assays have been the only reliable means of measuring enzyme activity. For kinases most of these methods use the phosphorylation of kinase substrates by cellular extracts or isolated proteins to estimate in vivo kinase activity (FIG. 3). There are three major drawbacks to this approach: 1) the methods have poor sensitivity so that the cytoplasm of large numbers of cells must be pooled; 2) a time-averaged level of the kinase activity is actually measured since the cells are not synchronous with respect to their activation status; and 3) the normal relationships of the pathways and networks within which signal transduction enzymes reside are disrupted. Similar drawbacks exist for measurement of the activity of most other types of enzymes. Recent imaging methods enable some enzymes to be spatially localized in single cells (FIG. 4). Generally the enzyme of interest is labeled within the cell by specific antibodies, inhibitors of the enzyme, or fluorescent tags. Unfortunately, both inactive and active enzyme molecules are highlighted by this method, although some attempt may be made to infer the activity level of the enzyme from its location within the cell. A third strategy still under development is the use of a fluorescent indicator to measure enzyme activity. This strategy has worked well for the measurement of various ion concentrations (i.e., Ca2+), but thus far has not been generally applicable to the measurement of enzyme activity in the living cell. A further limitation to all of these techniques is their inability to measure multiple enzyme activities simultaneously in a cell.